Hirudin is an anti-coagulation factor secreted from the salivary glands of medicinal leeches, Hirudo medicinalis, and is a mixture of peptides consisting of 65 and 66 amino acids. The structure of hirudin was determined by Dodt et al. [FEBS Lett. 165, 180(1984)] as hirudin variant 1 (HV1). Another variant, hirudin HV2, [Harvey et al., Proc. Natl. Acad. Sci. U.S.A., 83, 1084(1986)] has nine different amino acids in comparison with hirudin HV1, and still another variant, hirudin HV3, [Dodt et al., Biol. Chem. Hoppe-Seyler, 367, 803(1986)] has ten different amino acids in comparison with HV2, having the same sequence as HV2 up to Ser.sup.32, and inserting an amino acid (Ala.sup.63) in the C-terminal region. The structures of these three hirudin variants are shown in FIG. 1.
These natural type variants comprise 65 or 66 amino acids, and two domains are discernible. These are the spherical structured N-terminal region with three disulfide bonds, and the acidic C-terminal region exhibiting homology with the thrombin cleaving region of the pro-thrombin molecule, or with the fibrinogen cleaving region.
The present inventors have discovered that HV1 contains Leu.sup.64 -Gln.sup.65, while HV3 contains Asp.sup.65 -Glu.sup.66 in the C-terminal amino acid region. A patent application has been filed (Japanese Laid Open Patent Publication 3-164184 (1991)) on the synthesis of synthetic genes for HV1 and HV3 and their expression in E. coli.
Hirudin variants HV1, HV2 and HV3, having anti-thrombotic activity, are not acceptable drugs for clinical use because of their serious side effects, such as prolongation of bleeding time.
Several systems for producing hirudin by genetic engineering technology have been proposed; however, a satisfactory method has not yet been developed. From an industrial point of view, an extracellular production system is particularly desirable, since if the produced protein can be secreted extracellularly, there will be advantages not only simplifying the separation and purification of the product because of its presence in active form, but also because the product will be protected from digestion by intracellular bacterial proteases.
Methods using Bacillus subtilis, yeast or E. coli as hosts have been proposed for the production of hirudin by secretion.
Methods using Bacillus subtilis as host are disadvantageous in that plasmids are generally unstable in this bacterium, resulting in frequent curing of plasmids, making the stable protein production difficult, or the protein products in the medium are likely to be digested by their own proteases also secreted into the medium. Methods proposed for hirudin production (for example Japanese Laid Open Patent Publication 2-35084 (1990)) have not solved such problems, and the production yield is only about 100 mg/L.
In methods using yeast as hosts, it is known that the C-terminal amino acids of the products are hydrolyzed by carboxypeptidase.
In a prior report (N. Riehl-Bellon et al., Biochemistry 1989, 28, 2941-2949), by-products having 1 or 2 amino acid residues hydrolyzed off from the C-terminal end of HV2 are disclosed.
To overcome this problem, a method using carboxypeptidase-deficient yeasts (Japanese Laid Open Patent Publication 2-104279 (1990)) as hosts has been proposed, but has not led to sufficient productivity.
Using E. coli as host, a method using the alkaline phosphatase signal sequence has been reported (J. Dodt et al., FEBS Lett. 202, 373-377 (1986)). Although this is a secretion system, the product is secreted mainly into the periplasmic space, and is not satisfactory since it requires the disruption of bacterial cells by an additional recovery process step such as osmotic shock, and the product yield is as low as 4 mg/l.